Project Summary System-wide analysis of cell types in the brain is essential for understanding how complex cellular interactions give rise to various brain functions. Rapidly evolving tissue transformation and clearing technologies have enabled three-dimensional (3D) imaging and phenotyping of intact brains at unprecedented resolution. In particular, proteomic imaging techniques can provide multiscale anatomical, morphological, molecular, and functional information in both non-human and human brains. Highly specific antibodies, once rigorously validated, should enable spatial mapping of the brain proteome and corresponding cellular architectures; however, the unique potential of the 3D proteomic imaging is fundamentally limited by (1) incompatibility of existing antibodies with the emerging tissue transformation approaches, (2) poor specificity of commercially available antibodies, (3) lack of antibodies for newly identified targets, and (4) prohibitively high antibody cost for brain-wide labeling applications. The goal of this proposal is to democratize the 3D proteomic imaging approaches by creating a comprehensive open-source library of high-quality monoclonal antibodies (mAbs) that are truly renewable, scalable, and compatible with a wide range of conventional and emerging tissue processing technologies. In addition, we will develop robust and scalable protocols for uniform staining of intact brain tissues with new mAb tools. We envision that this antibody library and protocols will allow the research community to interrogate brain structure and function, including complex intercellular relationships at multiple scales. In Aim 1, we will create up to 3000 monospecific mAbs against 300 proteins of interest for BICCN brain mapping, general neuroscience research, and beyond. All mAbs will be screened for target protein binding specificity by using a combination of HuProt and VirD arrays. In Aim 2, we will extensively validate the IHC performance of the 3000 mAbs in a method-specific manner following the recommendations of the International Working Group for Antibody Validation (IWGAV). In Aim 3, we will create a library of renewable and cost-effective recombinant antibodies based on the validated mAbs from Aim 2. To immediately put the useful antibody toolbox in the hands of the neuroscience community, we will develop passive and cost-effective protocols for staining intact brain tissues with mAbs targeting key cell-types and activity markers. All validated antibodies, sequences, and staining protocols will be made available to the public.